The melanocyte can give rise to a number of morphologically different tumors. Most of them are biologically benign and are referred to as melanocytic nevi. Examples of melanocytic nevi are congenital nevi, Spitz nevi (including pigmented spindle cell nevi, which are regarded as a subtype of Spitz nevi), dysplastic or Clark""s nevi, blue nevi, lentigo simplex, and deep penetrating nevus.
Patients with congenital melanocytic nevi (CMN) have an increased risk of developing melanoma. Whereas in smallxe2x80x94( less than 1.5 cm) and intermediate-sized CMN (1.5-20 cm) the risk seems to be low (Rhodes, A. R., Med Clin North Am., 70:3-37 (1986); Sahin, S. et al., J Am Acad Dermatol., 39:428-33 (1998)), large CMN ( greater than 20 cm) carry a 5-15x times increased future risk to develop melanoma and rarely, other neural crest derived malignancies ((Swerdlow, A. J. et al., J Am Acad Dermatol., 32:595-9 (1995); Ruiz-Maldonado, R. et al., J Pediatr., 120:906-11 (1992); Quaba, A. A. and Wallace, A. F., Plast Reconstr Surg., 78:174-81 (1986); Gari, L. M. et al., Pediatr Dermatol., 5:151-8 (1988); Egan, C. L. et al., J Am Acad Dermatol., 39:923-32 (1998); Bittencourt, F. V. et al., Pediatrics, 106:736-41 (2000); DeDavid, M. et al., J Am Acad Dermatol., 36:409-16 (1997); Marghoob, A. A. et al., Arch Dermatol., 132:170-5 (1996)).
Melanoma refers to malignant neoplasms of melanocytes. Its proper diagnosis and early treatment is of great importance because advanced melanoma has a poor prognosis, but most melanomas are curable if excised in their early stages. Although, in general the histopathological diagnosis of melanoma is straightforward, there is a subset of cases in that it is difficult to differentiate melanomas from benign neoplasms of melanocytes, which have many variants that share some features of melanomas (LeBoit, P. E. Stimulants of Malignant Melanoma: A Rogue""s Gallery of Melanocytic and Non-Melanocytic Imposters, In Malignant Melanoma and Melanocytic Neoplasms, P. E. Leboit, ed. (Philadelphia: Hanley and Belfus), pp. 195-258 (1994)). Even though the diagnostic criteria for separating the many simulators of melanoma are constantly refined, a subset of cases remains, where an unambiguous diagnosis cannot be reached (Farmer et al., Discordance in the Histopathologic Diagnosis of Melanoma and Melanocytic Nevi Between Expert Pathologists, Human Pathol. 27: 528-31 (1996)).
During the neonatal period several types of melanocytic tumors can develop within CMN, many of which are thought to be distinct from melanoma (DeDavid, M. et al., J Am Acad Dermatol., 36:409-16 (1997)). These can be small to large, occasionally involving up to 50% of the trunk, and can grow very fast and ulcerate (Clark, W. H. et al., Pathology of the Skin, 1st edition, pp. 729-35, New York, McGraw-Hill (1990)). Most of these tumors have a benign course and tend to regress after a period of rapid growth. However, because true melanoma can occur in the neonate, the development of any secondary proliferation in a CMN is of significant concern.
These lesions can be extremely difficult to classify histologically. Four different histological patterns of proliferations in CMN during the neonatal period have been described (Clark, W. H. et al., Pathology of the Skin, 1st edition, pp. 729-35, New York, McGraw-Hill (1990)): 1) simulants of superficial spreading melanoma, in which the epidermis and superficial dermis contain large epitheloid melanocytes, sometimes with pagetoid spread in the epidermis; 2) simulants of nodular melanoma with a nodular proliferation of large melanocytes with uniform nuclei in the dermis; 3) cases described as xe2x80x9cproliferative neurocristic hamartomaxe2x80x9d, characterized by a deep dermal or subcutaneous proliferation with a variety of forms of neural or mesenchymal differentiation; and 4) true melanoma, most of which show small blast-like melanocytes with hyperchromatic nuclei, scant cytoplasm and a high mitotic rate.
The current invention is based on the discovery that chromosomal aberrations are common in atypical nodular proliferations and further, are absent from conventional congenital nevi. The aberrations are predominantly numerical changes, in contrast to those identified in melanoma, in which structural chromosomal aberrations are found in the vast majority of cases. These findings are useful in the classification of histopathologically ambiguous cases.
The current invention provides a method of typing a growth arising in association with a congenital nevus, the method comprising providing a skin tumor sample from a patient and detecting a change in chromosome number in a nucleic acid sample from the skin tumor sample. The change in chromosome number is typically a gain of chromosome 10, a gain of chromosome 11, or a loss of chromosome 7. The presence of one or more of these changes in the skin tumor sample types a lesion as a benign growth. Similarly, the absence of changes in chromosomal aberrations that are frequently associated with melanoma, types the skin tumor sample as a benign growth.
In one embodiment, the detection step comprises contacting a nucleic acid sample from the patient with a probe which selectively hybridizes to a target polynucleotide sequence on a chromosome selected from the group consisting of chromosome 10, chromosome 11, and chromosome 7; wherein the probe is contacted with the sample under conditions in which the probe binds selectively with the target polynucleotide sequence to form a stable hybridization complex; detecting the formation of the hybridization complex; and detecting a change in chromosome number, the change selected from the group consisting of a gain of chromosome 10, a gain of chromosome 11 and a loss of chromosome 7.
The nucleic acid sample can be, for example, an interphase nucleus or a metaphase cell. The probe is often labeled with a fluorescent labeled, but can also be labeled with other labels such as digoxigenin or biotin. Often, the probe is bound to a solid substrate and further, can be a member of an array. In some embodiments, the probe is a centromeric probe.
The methods of the invention can also comprise a further step of contacting the nucleic acid sample with a reference probe that binds selectively to a chromosome that does not undergo changes in chromosome number in these lesions. Such reference probes include, e.g., probes to chromosomes 1, 2, 4, 12, 13, 14, and 19.
Additionally, the methods of the invention can comprise a step of blocking the hybridization capacity of repetitive sequences in the nucleic acid sample. For example, unlabeled blocking nucleic acids such as comprising repetitive sequences, such as Cot-1 DNA, are contacted with the sample.
Definitions
To facilitate understanding the invention, a number of terms are defined below.
The terms xe2x80x9cmelanomaxe2x80x9d or xe2x80x9ccutaneous melanomaxe2x80x9d refer to malignant neoplasms of melanocytes, which are pigment cells present normally in the epidermis and sometimes in the dermis. There are four types of cutaneous melanoma: lentigo maligna melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral lentiginous melanoma (AM). Melanoma usually starts as a proliferation of single melanocytes at the junction of the epidermis and the dermis. The cells first grow in a horizontal manner and settle an area of the skin that can vary from a few millimeters to several centimeters. As noted above, in most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician.
The term xe2x80x9cmelanocytic neoplasmxe2x80x9d refers to an accumulation of melanocytes that can undergo a benign, locally aggressive, or malignant course. xe2x80x9cMelanocytic neoplasmxe2x80x9d encompasses both benign melanocytic neoplasms, xe2x80x9cnevixe2x80x9d, and malignant melanocytic neoplasms, xe2x80x9cmelanomaxe2x80x9d.
xe2x80x9cCongenital melanocytic nevixe2x80x9d refer to moles that are present at birth or arise shortly thereafter. Often, such nevi can grow rapidly. Rapidly growing nodules occasionally arise in these nevi during the neonatal period. Histologically, the nodules often present as atypical nodular proliferation of high cellularity, nuclear atypia and demonstrate and increased proliferation rate. A xe2x80x9cgrowth arising from a congenital nevusxe2x80x9d refers to an area of proliferation in a nevus. Using current histological criteria it is often difficult or impossible to classify these growth as either benign or malignant.
The terms xe2x80x9ctumorxe2x80x9d or xe2x80x9ccancerxe2x80x9d in an animal refers to the presence of cells possessing characteristics such as atypical growth or morphology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal. xe2x80x9cTumorxe2x80x9d includes both benign and malignant neoplasms.
As used herein, a xe2x80x9cnumerical aberrationxe2x80x9d refers to a change in number of a whole chromosome compared to normal, e.g., a gain of chromosome 10 refers to a gain of the entire chromosome. A xe2x80x9cstructural aberrationxe2x80x9d refers to a change in the structure of a chromosome. xe2x80x9cStructural aberrationsxe2x80x9d includes changes in copy number of chromosomal regions, e.g., changes in copy number of a chromosomal arm or subregions of a chromosomal arm, as well as chromosomal rearrangements such as translocations, insertions, deletions, and other rearrangements.
The phrase xe2x80x9ctypingxe2x80x9d or xe2x80x9cdetectingxe2x80x9d a neoplasm refers to the determination whether the neoplasm is, or has a high probability of being, a certain class of neoplasm. Classification can be based on whether the neoplasm is benign or malignant, or type of nevus, e.g., a congenital nevus. xe2x80x9cTypingxe2x80x9d or xe2x80x9cdetectingxe2x80x9d can also refer to obtaining indirect evidence regarding the likelihood of the presence of a benign growth or melanoma in the patient. Detection of a benign growth versus a melanoma can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the patient.
The terms xe2x80x9chybridizing specifically toxe2x80x9d, xe2x80x9cspecific hybridizationxe2x80x9d, and xe2x80x9cselectively hybridize to,xe2x80x9d as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. A xe2x80x9cstringent hybridizationxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I, Ch. 2, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assays,xe2x80x9d Elsevier, N.Y. (xe2x80x9cTijssenxe2x80x9d). Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42xc2x0 C. using standard hybridization solutions (see, e.g., Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, e.g., Sambrook supra.) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4xc3x97 to 6xc3x97SSC at 40xc2x0 C. for 15 minutes.
The term xe2x80x9clabeled with a detectable labelxe2x80x9d, as used herein, refers to a nucleic acid attached to a detectable composition, i.e., a label. The detection can be by, e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or chemical means. For example, useful labels include 32P, 35S, 3H, 14C, 125I, 131I; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense reagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. Dynabeads(trademark)), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label can be directly incorporated into the nucleic acid, peptide or other target compound to be detected, or it can be attached to a probe or antibody that hybridizes or binds to the target. Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield, Mol Cell Probes 9: 145-156 (1995). In addition, target DNA sequences can be detected by means of the primed in situ labeling technique (PRINS) (Koch et al., Genet. Anal. Tech. Appl. 8: 171-8, (1991)). The sensitivity of the detection can be increased by using chemical amplification procedures, e.g., by using tyramide (Speel et al., J. Histochem. Cytochem. 45:1439-46, (1997)).
The term xe2x80x9cnucleic acidxe2x80x9d as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
The term a xe2x80x9cnucleic acid arrayxe2x80x9d as used herein is a plurality of target elements, each target element comprising one or more nucleic acid molecules (probes) immobilized on one or more solid surfaces to which sample nucleic acids can be hybridized. The nucleic acids of a target element can contain sequence(s) from specific genes or clones, e.g. from specific regions of chromosomes 7, 10, or 11. Other target elements will contain, for instance, reference sequences. Target elements of various dimensions can be used in the arrays of the invention. Generally, smaller, target elements are preferred. Typically, a target element will be less than about 1 cm in diameter. Generally element sizes are from 1 xcexcm to about 3 mm, preferably between about 5 xcexcm and about 1 mm. The target elements of the arrays may be arranged on the solid surface at different densities. The target element densities will depend upon a number of factors, such as the nature of the label, the solid support, and the like. One of skill will recognize that each target element may comprise a mixture of nucleic acids of different lengths and sequences. Thus, for example, a target element may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths. The length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure, and to provide the required resolution among different genes or genomic locations.
The terms xe2x80x9cnucleic acid samplexe2x80x9d or xe2x80x9csample of human nucleic acidxe2x80x9d as used herein refers to a sample comprising human DNA or RNA in a form suitable for detection by hybridization or amplification. Typically, it will be prepared from a skin tissue sample of a tumor from a patient who has or is suspected of having a melanocytic tumor that may be difficult to classify.
The nucleic acid sample is often be a tissue or cell sample prepared for hybridization using methods described below. The sample is prepared such that individual chromosomes remain substantially intact according to standard techniques. Alternatively, the nucleic acid may be isolated, cloned or amplified. It may be, e.g., genomic DNA, mRNA, or cDNA from a particular chromosome, or selected sequences (e.g. particular promoters, genes, amplification or restriction fragments, cDNA, etc.) within particular amplicons or deletions disclosed here. In some cases, the nucleic acids may be amplified using standard techniques such as PCR. The sample may be isolated nucleic acids immobilized on a solid.
The nucleic acid sample is typically extracted from particular cells, e.g. melanocytes, or prepared from a skin tumor, i. e., a melanocytic neoplasm. Methods of isolating cell and tissue samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, needle biopsies, and the like. Frequently the sample will be a xe2x80x9cclinical samplexe2x80x9d which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes. The sample can also be derived from extracts or supernatants from the cells or the cells themselves from cell cultures, cells from tissue culture and other media in which it may be desirable to detect chromosomal abnormalities, such as changes in copy number. In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample may be isolated nucleic acids immobilized on a solid.
The term xe2x80x9cprobexe2x80x9d or xe2x80x9cnucleic acid probexe2x80x9d, as used herein, is defined to be a collection of one or more nucleic acid fragments whose specific hybridization to a sample can be detected. The probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the regions described herein. Often, the probes are centromeric probes, i.e., they hybridize to nucleic acid sequences present in the centromeres of the specific chromosomes, which provide a stronger signal.
The probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. The word xe2x80x9csamplexe2x80x9d may be used herein to refer not only to detected nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, e.g., with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately.
What xe2x80x9cprobexe2x80x9d refers to specifically is clear from the context in which the word is used. The probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. In some embodiments, the probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854). One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are xe2x80x9csubstantially identicalxe2x80x9d to the disclosed probes, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived (see discussion above). Such modifications are specifically covered by reference to the individual probes described herein.
A xe2x80x9creference probexe2x80x9d refers to a control probe that specifically hybridizes to a chromosome that does not undergo changes in number in growths that arise from congenital nevi.
xe2x80x9cProviding a nucleic acid samplexe2x80x9d means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g. isolated by another person).
xe2x80x9cTissue biopsyxe2x80x9d refers to the removal of a biological sample for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.